Modification, Selection and Production of Cyclic Peptides Nicole Albrecht1, Katharina Berger1, Nadja Bjelopoljak1, Nadine Böhmer1, Jessica Eger1, Sebastian Hanke1, Sandrina Heyde1, Paul Kaufmann1, Niklas Laasch1, Sabine Meyer1, Sascha Ramm1, Stefanie Sempert1,

Stefan Wahlefeld1, Niels Weisbach1, Tobias Wenzel1,2, Oliver Zimmer1,3, Tobias Baumann1, Sven Hagen1, Tim Kükenshöner1, Katja Arndt1 & Kristian Müller1 1) iGEM Team Potsdam, Universität Potsdam, Karl-Liebknechstr. 24 - 25, 14476 Potsdam Golm, kristian@syntbio.net

2) Technische Universität Berlin 3) TH Wildau

One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Towards this goal, we mutate and select microviridins, which are tricyclic depsipeptides from cyanobacteria. They are small but stable due to their post-translational side-chain cross linking. …………

Abstract mdnA Library

V = A/C/G H = A/C/T K = G/T N = A/C/G/T

YGGTFKYPSDWEDY

Leader peptide Core peptide

THT GVT GVT ACC NKK AAA TAC CCT TCT GAC TGG GAA GAT THT 3 3 3 1 10 1 1 1 1 1 1 1 1 3 810

MAYPNDQQGKALPFFARFLSVSKEESSIKSPSPEPT

Figure 5: Design and confirmation of our mdnA library. The precursor peptide (mdnA) of microviridin consists of a leader and core peptide. To find optimized microviridin variants the mdnA core peptide was synthesized using specifically randomized oligonucleotides. We generated several libraries with various diversities, the smallest (diversity of 810) is shown and the modifications are highlighted (A). Sequencing of 10 clones revealed the planned diversity (B) and the library is ready for screen against (therapeutic) protease targets.

A

B

Characterization of mdnA

Sonification

C18 Sep-Pak cartridges

HPLC

Mass Spectrometry

exact mass of bicyclic microviridin

Expression of microviridin in E. coli

Figure 4: Mass spectrometry analysis of microviridin separated by HPLC.

3 8 13 18Time in minutes

Figure 3: Reversed phase HPLC analysis of microviridin produced by pARW089.

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Sponsors & References

Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007). Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-7

Fuh G., Sidhu S.S. (2000). Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein. FEBS Lett. 480(2-3):231-4

Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000.

Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Meth. 201(1):35-55

Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30: 544-550.

Olivier Genest, Marianne Ilbert, Vincent Méjean and Chantal Iobbi-Nivol April 22, 2005 The Journal of Biological Chemistry, 280, 15644-15648

Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95

Rakonjac J., Feng J., Model P. (1999). Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII. J Mol Biol. 289(5):1253-65

Smith, G.P. (1985). Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virus surface. Science 228: 1315-17 Waraho, D. & DeLisa, M. P. (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7 Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74

Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9

Human Practice & Safety

We conducted a survey on the opinion of the 622 members of the German parliament relating to the potential and risk of Synthetic Biology in food, energy and healthcare. Additionally, we discussed safety issues according to the German ‘Law on Gene Technology’, thereby minimizing the danger for the environment and our team.

Figure 13: Answer to question: ”How would you assess the potential of Synthetic Biology?” (A) and answer to question: ”How would you assess the risk of Synthetic Biology?” (B). (C) Children assisting us in the lab and

enjoying tasks like pipetting. (D) Our team visiting the German parliament.

70%

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High Medium Low Abstention

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D

Expression Backbones

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Time in min

--- pSB1A3_Ara --- control

Figure 16: Emission (526 nm) of E. coli cells carrying pSB1A3 with an arabinose-induced promoter and yfp as reporter gene compared to control cells with noninduced promoter.

Figure 15: Fluorescence microscopy pictures of E. coli carrying an arabinose-inducible promoter and the yfp gene and control cells.

YFP DIC

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BBa_K627015

Amp

YFP Ara EcoRI XbaI SpeI PstI

Figure 14: Scheme of our constructed expression backbone. This contains an arabinose-inducible promoter (Ara) and yfp as reporter gene.

Using our auxiliary expression backbones you can replace the reporter gene by your gene of interest via the iGEM restriction sites.

Conclusion

In Vivo Selection

Our in vivo system selects inexpensively, time-saving and efficiently inhibitor libraries for proteases. For coupling of protease activity to cell survival a detection device was constructed, in which antibiotic resistance conferring beta-lactamase is fused to TorA via protease cleavage site. TorA addresses the TAT pathway and, thus, requires time for folding and provides protease access. Cleavage by the protease blocks translocation and, thus, abolishes resistance. Inhibition of the protease by a microviridin variant enables transport and antibiotic resistance. Ultimately, our generation and detection system in combination with our library yielded microviridin clones.

Amplification of parts

Ligation in pSB1C3

Combination

Bba_K627008

AraC Protease

Bba_K627012

TorA Protease

cleavage site β-lactamase

Experimental data: figure 11A

pUP_SG1

TorA

AraC

Protease cleavage site

Cm Protease

β-lactamase

Experimental data: figure 11B

Perfect working selection vector (protease generator and activity detector)

Double transformation with mdnA library

Selection for optimized microviridin

Figure 12: Schematic construction and principle of our selection vector containg protease generator and protease activity detector.

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Ampicillin concentration in µg/ml

TEV protease not induced TEV protease induced

Figure 11: Survival tests of E. coli cells carrying the complete protease activity detector with TEV cleavage site (Figure 12) at several ampicillin concentrations. A: Induced ß-lactamase, protease not induced. B: Induced ß-lactamase and the protease induced by arabinose (magenta coloured bars). C: Vector which contains the protease activity detector device and the protease generator device (Figure 12) with induced ß-lactamase and TEV protease. The β-lactamase (bla) was induced by IPTG and the TEV protease was induced by arabinose. All plates contained chloramphenicol. Y-axis shows percentage of surviving cells.

Microviridins have a high potential for therapy as they can block disease-relevant proteases. Yet, the possibilities of cyclic peptides are largely untapped since genetic systems for optimization are not well established. Thus, we developed synthetic systems for the mutation, selection and production of such peptides. We utilized the 6.5 kb microviridin (mdn) gene cluster cloned in E. coli plasmids, established random mdnA mutagenesis and generated focused libraries of microviridins. For selection against a panel of proteases, we are applying and testing phage display, and we are constructing a novel in-vivo selection device, which links protease blocking to antibiotic resistance. Our systems adhere to the BioBrick standards.

Selected microviridin clones

A

B

C

In vitro Selection by Phage Display

For screening of our mdnA libraries, we established an in vitro selection method based on phage display. We generated a phagemid vector carrying myc-tag and the short gene III inserted in the microviridin gene cluster (mdnABCDE,). Successful expression was demon-strated by western blot and display on phages by ELISA using anti-myc antibodies. The mdnA phages were panned against the protease trypsin which is a known target of mdnA. After one panning round mdnA displaying phages were enriched over the control. Further, binding of other proteases was tested.

Figure 8: Detection of phages carrying MdnA on their surface using ELISA. Immobilized anti-myc antibodies and a second antibody coupled with HRP were used.

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Figure 9: Optimization of phage display conditions. Enrichment of phages displaying MdnA from a phage mix in a 1:1 ratio after one panning round was achieved.

Figure 6: Our constructed phagemid vector containing myc, the short gene III and microviridin gene cluster (mdnABCDE), which was used for our phage display experiments.

pPDV089

mdnD mdnB myc gene III mdnA

Confirmation of mdnA-myc-gene III expression

Experimental data: figure 7

Generation of phagemid vector

Confirmation of mdnA presentation on the

surface of the phages

Screening for optimized microviridins

Examine phage displays suitability as selection

method (wt microviridin)

See figure 6

Experimental data: figure 8

Experimental data: figure 9

Testing phage display against several proteases

(wt microviridin) Experimental data: figure 10

mdnE mdnC

mdnA-myc- geneIII protein cells marker

30 kDa

Figure 7: Control of mdnA-myc-geneIII expression in E. coli by western blot. For detection anti-myc-antibodies and secondary HRP-linked antibodies were used.

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Figure 10: Phage display against different proteases. The interaction of microviridin with chymotrypsin and elastase was confirmed and an interaction with papain was noted.

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Microcystis aeruginosa

Mutagenesis System

mdn cluster

Protease Blocking Detector by Cell Survival

M13 Phage

Pili E. coli

Protease Target Binding Selector by Phage Display

Inducible Microviridin Production System

Optimized Protease Inhibitor

Protease

mdnA

TorA-β-lactamase

Expression Backbones

mdnA- myc- geneIII

Periplasm

β-lactamase

Signal sequence TorA

Protease

Microviridin

Our generation and detection system in combination with our library yielded microviridin clones. This shows that our in vivo selection system works as expected. Additionally, we confirmed that our in vitro selection system is also well working.

Further we designed and generated 13 BioBricks

5 for mdnA modularization 6 for in vivo selection 2 for phage display

We conducted a survey on the opinion of the 622 members of the German parliament. Also we discussed with one representative about Synthetic Biology.

Modularization of the mdn Cluster

Using two vectors containing the mdn biosynthesis gene cluster, we obtained the gene fragments of mdnA, mdnB, mdnC, mdnD and mdnE. Out of these genes five different BioBricks were built.

Figure 2: Modularization of the mdn gene cluster (mdnABCDE) encoding the tricyclic peptide microviridin and all enzymes, which are essential for correct processing.

Precursor peptide

Ligase Ligase N-Acetyl-transferase

BBa_K627005

mdnE

BBa_K627004

mdnD

BBa_K627002

mdnB

BBa_K627001

mdnA

BBa_K627003

mdnC

pARW089

mdnB mdnA mdnD mdnE mdnC

ABC Transporter

Microviridin and Therapy

Linear peptides are typically unstable within the cell and are rapidly degraded by proteases. In contrast cyclic peptides exhibit a higher stability and therefore have a great therapeutic potential. Our team was very interested in the tricyclic depsipeptide microviridin, which is produced by cyano- .

Figure 1: Correctly processed microviridin built up from the precursor peptide (mdnA).

bacteria. Their special ability is to inhibit several proteases. Microviridins are built up by a gene cluster (mdnABCDE, Figure 2), which is essential for the correct processing of the precursor peptide mdnA to microviridin. Its unusual cage-like architecture is due to the ω-amide bonds.

Modeling of In Vivo Selection

Our systems modeling analyzes the reaction kinetics of the in vivo selection system and predicts the outcomes. The simulation is grouped in three time frames:

1) expression of the protease 2) expression of TorA-cleavage-site-β-lactamase 3) antibiotic selection

The analysis of the mathematical model shows that the system is stable in and around the predicted order of magnitudes of constants and initial conditions. Variations of expression para-meters and interaction constants allow the estimation of optimal selection conditions.

Figure 18: Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient KD.

Figure 17: Comparison of model and measurement at t 4 > 3h. Percentage of surviving cells dependent on the ampicillin concentration added to the medium.